NEAT1/microRNA 339-5p/SPI1 Axis Feedback Loop Contributes to Osteogenic Differentiation in Acute Suppurative Osteomyelitis in Children

Abstract

Objective: Long non-coding RNA plays an important role in osteogenic differentiation. Nuclear enriched abundant transcript 1 (NEAT1) has been revealed to promote osteogenic differentiation in human bone marrow mesenchymal stem cells (hBMSCs), but the underlying regulatory mechanism remains unknown in acute suppurative osteomyelitis of children.

Methods: Osteogenic medium (OM) was used to induce osteogenic differentiation. Quantitative real-time PCR and Western blotting were used to evaluate gene expression. The effects of NEAT1, microRNA 339-5p (miR-339-5p), and salmonella pathogenicity island 1 (SPI1) on osteogenic differentiation were assessed in vitro using alizarin red S staining assays and alkaline phosphatase activity. Interactions between NEAT1, miR-339-5p, and SPI1 were identified using immunoprecipitation, luciferase reporter assays, and chromatin immunoprecipitation.

Results: During osteogenic differentiation, expression of NEAT1 was up-regulated in hBMSCs, and miR-339-5p level was down during osteogenic differentiation. Knockdown of NEAT1 reduced the osteogenic differentiation of hBMSCs, and down-regulation of miR-339-5p may counteract the effect of NEAT1 silencing. SPI1 was a target of miR-339-5p by luciferase reporter assay and was also a transcription factor of NEAT1 by chromatin immunoprecipitation. A positive NEAT1-miR-339-5p-SPI1 feedback loop was found to be present during osteogenic differentiation in hBMSCs.

Conclusion: It was the first study to reveal that the NEAT1-miR-339-5p-SPI1 feedback loop can promote osteogenic differentiation in hBMSCs and shed a new light on the role of NEAT1 during osteogenic differentiation.

Keywords: NEAT1; SPI1; miR-339-5p; osteogenic differentiation.

Figure 1

NEAT1 expression levels in osteomyelitis. (A) NEAT1 expression at mRNA level was lower in children with acute suppurative osteomyelitis; (B) Receiver operating characteristic curve of NEAT1 for acute suppurative osteomyelitis in children; (C) Relative mRNA levels of NEAT1 were examined by RT-qPCR assay in hBMSCs after 0, 3, 7, 14, and 21 days of incubation in an osteogenic medium (OM) relative to a growth medium (GM); (D) Relative mRNA levels of RUNX2 were examined by RT-qPCR assay in hBMSCs after 0, 3, 7, 14, and 21 days of incubation in OM relative to GM. (E) Relative protein levels of RUNX2 were examined by Western blotting assay in hBMSCs after 0, 3, 7, 14, and 21 days of incubation in OM relative to GM. (F and G) Relative mRNA levels of OSX and ALPLwere examined by RT-qPCR assay in hBMSCs after 0, 3, 7, 14, and 21 days of incubation in OM relative to GM.*P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

Knockdown of NEAT1 inhibits the mineralization ability of hBMSCs. (A) RT-qPCR analysis of the transfection efficiency of si-NEAT1 in hBMSCs. (B) RUNX2 mRNA levels were detected by RT-qPCR assay in hBMSCs after NEAT1 knockdown; (C) Relative protein levels of RUNX2 were examined by Western blotting assay in hBMSCs after NEAT1 knockdown; (D and E) ALPL and OSX mRNA levels were detected by RT-qPCR assay in hBMSCs after NEAT1knocdown; (F) ALP activity assays were conducted to measure the ALP activity upon the knockdown of NEAT1 in hBMSCs after osteogenic transduction; (G) ARS staining assays were performed on hBMSCs after osteogenic induction to examine the effect of mineralized bone matrix formation on ectopic NEAT1 expression. ***P < 0.001.

Figure 3

NEAT1 could inhibit miR-339-5p through TDMD mechanism. (A) NEAT1 was found to have different cellular sublocalizations in different cell lines via the lncLocator website: lncATLAS; (B) The localization of NEAT1 in hBMSCs was identified by subcellular fractionation assay and qRT-PCR; (C) FISH revealed that NEAT1 is highly expressed in the cytoplasm; (D) Potential miRs capable of binding to NEAT1 were predicted by using starBase; (E) Correlation between expressions of NEAT1 and miR-339-5p in children with acute suppurative osteomyelitis; (F) Relative mRNA levels of miR-339-5p were examined by RT-qPCR assay in hBMSCs after 0, 3, 7, 14, and 21 days of incubation in an osteogenic medium (OM) relative to a growth medium (GM); (G) The levels of miR-339-5p in hBMSCs after NEAT1 knockdown were detected by RT-qPCR assay; (H) The binding sites of lncRNA NEAT1 and miR-339-5p; (I) RNA pulldown assays were performed to confirm the physical interaction between NEAT1 and miR-339-5p in hBMSCs; (J) An RIP assay was performed to confirm the association of NEAT1 with miR-339-5p. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

NEAT1 negatively regulated miR-339-5p to inhibit osteogenic differentiation in vitro. (A) miR‑339‑5p expression in hBMSCs transfected with miR mimics and miR inhibitor; (B) RUNX2 mRNA levels were detected by qPCR after transfected with miR‑339‑5p mimics or inhibitor in hBMSCs; (C)Relative protein levels of RUNX2 were examined by Western blotting assay after transfected with miR‑339‑5p mimics or inhibitor in hBMSCs; (D and E) ALPL and OSX mRNA levels were detected by qPCR after transfected with miR‑339‑5p mimics or inhibitor in hBMSCs; (F) RUNX2 mRNA expression was determined by RT-qPCR assay in hBMSCs transfected with si-NC, si-NEAT1, si-NEAT1 plus NC inhibitor and si-NEAT1 plus miR-339-5p inhibitor after osteogenic induction; (G) RUNX2 protein levels were determined by Western blotting assay in hBMSCs transfected with si-NC, si-NEAT1, si-NEAT1 plus NC inhibitor and si-NEAT1 plus miR-339-5p inhibitor after osteogenic induction; (H and I) ALPL and OSX mRNA expressions were determined by RT-qPCR assay in hBMSCs transfected with si-NC, si-NEAT1, si-NEAT1 plus NC inhibitor and si-NEAT1 plus miR-339-5p inhibitor after osteogenic induction; (J) ALP activity assays were carried after osteogenic transduction under different transfection conditions; (K) ARS staining assays were performed following osteogenic transduction under different transfection conditions. ns, no significance, *P < 0.05,**P < 0.01, ***P < 0.001.

Figure 5

miR-339-5p can negatively regulate SPI1 in hBMSCs. (A) The putative binding sites between miR-339-5p and SPI1 were predicted by bioinformatics analysis; (B) miR-339-5p negatively regulated SPI1 in hBMSCs; (C) An RIP assay was conducted to confirm the association of miR-339-5p with SPI1; (D) The miR-339-5p was targeted at SPI1 3’UTR; (E) Luciferase reporter gene assays were performed to detect the fluorescence activity of the SPI1 3’UTR in hBMSCs co-transfected with wild-type SPI1 3’UTR or mutational type SPI1 3’UTR and miR-339-5p mimics/ inhibitors, respectively; (F) The SPI1 mRNA expression levels were detected by qRT-PCR in hBMSCs co-transfected with SPI1 siRNA and miR-339-5p inhibitor; (G) RUNX2 levels were detected by qRT-PCR in hBMSCs co-transfected with SPI1 siRNA and miR-339-5p inhibitor; (H) RUNX2 protein levels were determined by Western blotting assay in hBMSCs co-transfected with SPI1 siRNA and miR-339-5p inhibitor; (I and J) OSX and ALPL levels were detected by qRT-PCR in hBMSCs co-transfected with SPI1 siRNA and miR-339-5p inhibitor; (K) ALP activity assays were detected in hBMSCs co-transfected with SPI1 siRNA and miR-339-5p inhibitor; (L) ARS stainings were performed in hBMSCs co-transfected with SPI1 siRNA and miR-339-5p inhibitor. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

SPI1 formed a positive feedback loop with NEAT1/miR-339-5p/SPI1. (A) The binding motif of SPI1 was obtained from JASPAR; (B) The affinity of SPI1 in the promoter region of NEAT1 was assessed using ChIP assay; (C) Predicted binding sites of SPI1 in the promoter region NEAT1; (D) Luciferase reporter assay demonstrated the effect of SPI1 on the transcriptional activity of NEAT1; (E) Graphical summary of the NEAT1/miR-339-5p/SPI1 feedback loop in osteogenic differentiation. ns, no significance,***P < 0.001.